Assays for potency of human retinal pigment epithelium (RPE) cells and photoreceptor progenitors

ABSTRACT

This disclosure provides a new phagocytosis assay to test the function of RPE cells and photoreceptor progenitors using a pH sensitive fluorescent label.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/560,584, filed Sep. 22, 2017, entitled “IMPROVED ASSAYS FOR POTENCYOF HUMAN RETINAL PIGMENT EPITHELIUM (RPE) CELLS AND PHOTORECEPTORPROGENITORS”, which is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/US2016/023839, filed Mar. 23, 2016,which was published under PCT Article 21(2) in English, and which claimsthe benefit under 35 U.S.C. § 119(e) of U.S. Provisional ApplicationSer. No. 62/136,660, filed Mar. 23, 2015, entitled “IMPROVED ASSAYS FORPOTENCY OF HUMAN RPE CELLS AND PHOTORECEPTOR PROGENITORS”, the entirecontents of each of which are incorporated herein by reference.

FIELD OF INVENTION

This invention relates to the use of an in vitro cell-based method tomeasure phagocytosis of photoreceptor outer rod segments.

BACKGROUND

The retinal pigment epithelium (RPE) is the pigmented cell layer outsidethe neurosensory retina between the underlying choroid (the layer ofblood vessels behind the retina) and overlying retinal visual cells(e.g., photoreceptors—rods and cones). The RPE is critical to thefunction and health of photoreceptors and the retina. The RPE maintainsphotoreceptor function by recycling photopigments, delivering,metabolizing, and storing vitamin A, phagocytosing rod photoreceptorouter segments, transporting iron and small molecules between the retinaand choroid, maintaining Brach's membrane and absorbing stray light toallow better image resolution. See. e.g., WO 2009/051671; Engelmann andValtink (2004) “RPE Cell Cultivation.” Graefe's Archive for Clinical andExperimental Ophthalmology 242(1): 65-67; Irina Klimanskaya, RetinalPigment Epithelium Derived Prom Embryonic Steal Cells, in STEM CELLANTHOLOGY 335-346 (Bruce Carlson ed., 2009).

Degeneration of the RPE can cause retinal detachment, retinal dysplasia,or retinal atrophy that is associated with a number of vision-alteringailments that result in photoreceptor damage and blindness such aschoroideremia, diabetic retinopathy, macular degeneration (includingage-related macular degeneration. AMD) and Stargardt's macular dystrophy(SMD), the latter two being two of the leading causes of adult andjuvenile blindness in the world, respectively. Although both arecurrently untreatable, there is evidence in preclinical models ofmacular degeneration that transplantation of hESC-derived RPE can rescuephotoreceptors and prevent visual loss (Lund R D, Wang S, Klimanskaya I,et al. Human embryonic stem cell-derived cells rescue visual function indystrophic rats. Cloning and Stem Cells 2006; 8, 189-199; Lu B, MalcuitC, Wang S, et al. Long-term safety and function of RPE from humanembryonic stem cells in preclinical models of macular degeneration. StemCells 2009; 21, 2125-2135).

Also contributing to some of the afore-mentioned diseases is theadditional loss of post-mitotic neuronal cells. Among these retinaldiseases are rod or cone dystrophies, retinal degeneration, retinitispigmentosa (RP), diabetic retinopathy, macular degeneration, Lebercongenital amaurosis and Stargardt disease. In most cases of retinaldegeneration, cell loss is primarily in the outer nuclear layer (ONL)which includes rod and cone photoreceptors.

A potential replacement source of photoreceptor cells includes stemcells. Early studies evaluated mouse cells, mouse stem cells orheterogeneous populations of retinal progenitor cells as a possiblesource of replacement cells for lost photoreceptors. These early studiesdescribed transplantation of photoreceptor precursor cells frompostnatal day 1 mouse retina (Maclaren et al. Nature 444(9):203-207,2006), in vitro generation of retinal precursor cells from mouseembryonic stem cells (Ikeda et al. Proc. Natl. Acad. Sci.102(32):11331-11336, 2005), generation of retinal progenitor cells frompostnatal day 1 mouse retinas (Klassen et al. Invest. Ophthal. Vis, Sci.45(11):4167-4175, 2004), implantation of bone marrow mesenchymal stemcells in an RCS rat model of retinal degeneration (Inoue et al. Exp. EyeRes. 8(2):234-241, 2007), production of retinal progenitor cells,including ganglion cells, amacrine cells, photoreceptors wherein 0.01%of the total cells expressed S-opsin or rhodopsin, bipolar cells andhorizontal cells, from the H1 human embryonic stem cell line (Lamba etal. Proc. Natl. Acad. Sci, 10(34): 12769-12774, 2006), and induction ofinduced pluripotent stem cells (iPS) from human fibroblasts to produceretinal progenitor cells (Lamba et al. PLoS ONE 5(1):e8763.doi:10.1371/journal.pone.0008763). None of these approaches produced ahomogeneous population of photoreceptor progenitor cells orphotoreceptor cells for implantation. None of these approaches produceda homogeneous population of photoreceptor progenitor cells orphotoreceptor cells that showed in vivo rod or cone function (e.g.,detectable by conferring improvements in visual acuity). Supplies ofdonor-derived tissue from which photoreceptors and photoreceptorprogenitors may be isolated (such as cadavers, fetal tissue, and liveanimals) are limited. Stem cells can be propagated and expanded in vitroindefinitely, providing a potentially inexhaustible source of non-donorderived cells for human therapy. Differentiation of stem cells into ahomogeneous population of photoreceptor progenitors or photoreceptorsmay provide an abundant supply of non-donor derived cells forimplantation and treatment of retinal diseases. The photoreceptorprogenitor cells may have phagocytic activity.

Certain subject matter including methods of making RPE cells,compositions of RPE cells, and release assays (including phagocytosisassays) for RPE cells are disclosed in co-owned U.S. application Ser.No. 13/510,426, filed Nov. 17, 2010 and PCT US2012/65091, such teachingsincorporated by reference herein. Certain subject matter includingmethods of making photoreceptor progenitors, and compositions ofphotoreceptor progenitors and methods of testing function (includingphagocytosis assays) are disclosed in co-owned PCT US2014/029790, suchteachings incorporated by reference herein.

SUMMARY

FITC-labeled Photoreceptor Outer Segments (OS) (usually bovine orporcine) have been used to study phagocytosis by retina pigmentepithelium (RPE) in vitro. However, most of the quantitative methodsused for phagocytosis assessment (FACS, fluorescence plate reader) donot differentiate between surface-bound and internalized particles andthus do not allow one to specifically address mechanisms involved insurface receptor binding and internalization stages of phagocytosis.Additionally, FITC fluorescence is pH-sensitive and is significantlyreduced at pH below 6 while the pH of lysosomes and phagosomes fusedwith lysosomes is below 5. Thus fluorescence of FITC-labeled OS may notbe truly representative of the amount of internalized OS.

Provided herein is a more sensitive and accurate assay for detectinginternalized phagocytosis of photoreceptor outer segments, an importantmeasure of RPE cell and photoreceptor progenitor function andconsequently an important release criteria for RPE cells andphotoreceptor progenitors to be used in treating retinal diseases, suchas rod or cone dystrophies, retinal degeneration, retinitis pigmentosa,choroideremia, diabetic retinopathy, macular degeneration (includingage-related macular degeneration and myopic macular degeneration), Lebercongenital amaurosis and Stargardt disease (fundus flavimaculatus). See,e.g., WO 2009/051671.

The RPE cells described herein are functional after transplantation. Tothis end, the RPE cells form a monolayer between the neurosensory retinaand the choroid in the subject (or patient) receiving the transplantedcells. The RPE cells may also supply nutrients to adjacentphotoreceptors and dispose of shed photoreceptor outer segments byphagocytosis.

RPE cells suitable for transplantation may be selected based on a numberof functional and/or phenotypic characteristics, including but notlimited to phagocytosis activity. Four example, RPE cells suitable fortransplantation may be assessed according to their phagocytosis activityas well as their proliferative potential. For example, the RPE cells mayhave greater proliferative potential than cells derived from eye donors(e.g., the RPE cells are “younger” than those of eye donors). Thisallows the RPE cell described herein to have a longer useful lifespanthan cells derived from eye donors.

One of the key parameters to RPE cell potency in the clinical context isthe quantitative measure of the outer segment phagocytosis activity ofthe pharmaceutical preparations of RPE cells. The phagocytosis of theouter segments results in the accumulation of the phagocytosed cellfragments in low-pH compartments in the RPE cells. The present inventionprovides for photoreceptor outer segments that have been associated(i.e., covalently or non-covalently) with a detectable marker which isdetectable spectrophotometrically to provide a spectrophotometricsignal, the detectable marker being selective to have a firstspectrophotometric signal when present at a neutral pH or physiologicalpH, i.e., a pH of 7 to 7.5, and second spectrophotometric signal whenpresent in the low pH environment of an intracellular compartment, suchas a lysosome, phagosome, endosome, or the like. The difference betweenthe first and second spectrophotometric signals may be one or more ofthe degree of fluorescence emission (increased intensity at low pHrelative to neutral pH), a change in the fluorescence emissionwavelength between neutral and low pH, a change in the fluorescenceexcitation wavelength between neutral and low pH, or the like.

In certain embodiments, the detectable marker can be a fluorescent pHsensor, such as fluorescent dye. Exemplary fluorescent dyes that can beused in the instant invention may be a fluorescent dye moiety having anamino group (aliphatic or aromatic) as the pH sensitive indicatormoiety, i.e., an amine which is unprotonated at the pH of the culturemedia in which the RPE cells and outer segments are incubated together(i.e., a neutral pH or physiological pH), and becomes protonated at thepH of the intracellular compartment into which the outer segments areabsorbed by the cells such as the RPE cells by phagocytosis. When such adye adsorbs a photon, creating an excited electronic state, the electronof the amino group's unshared pair transfers to the orbital vacated byexcitation. Such an electron transfer, referred to as PhotoinducedElectron Transfer (PET) prevents the excited molecule from emissiontransition, thus the fluorescence of the dye is quenched. Protonation ofthe amino group changes the nature and energy of the pair's orbital andstops the PET. As a result, the fluorescent reporter moiety responds toa pill change. Because protonation of the amino group cancels thequenching, the PET-based sensors become more fluorescent as pHdecreases.

In certain embodiments, the fluorescent dye is a rhodamine-based pHsensitive dye, such as described in WO 2005/098437. These dyes have abenzene ring substituted ortho to the xanthene moiety by —OH or —SH (ortheir depronated forms). These dyes display a pH-dependency similar toamine PET indicators but were designed to have pKa values of less than 6based on a perceived need for a pH sensor that would target cellcompartments with a pH of less than 6.

In another exemplary embodiment, the fluorescent marker is apH-sensitive fluorescent nanoparticle. pH-sensitive fluorescentnanoparticles primarily employ polymers conjugated with small molecularpH-sensitive dyes (Srikun, D., J. Chem. Sci. 2011, 2, 1156; Benjaminsen,it V., ACS Nano 2011, 5, 5864; Albertazzi, L., J. Am. Chem. Soc. 2010,132, 18158; Urano, Y., Nat. Med. 2009, 15, 104) or the use ofpH-sensitive linkers to conjugate pH-insensitive dyes (Li, C, Adv. Fund.Mater. 2010, 20, 2222; Almutairi, J. Am. Chem. Soc. 2007, 130, 444). Tofurther illustrate, WO 2013152059 describes pH-tunable, highlyactivatable multicolored fluorescent nanoplatforms which can be adaptedfor use in the present assays.

Thus, provided herein in one aspect is a method for assessingphagocytosis activity comprising incubating cells with photoreceptorouter segments (POS) for a time and temperature sufficient for the cellsto phagocytose the POS, wherein the POS fluoresce more at an acidic pHthan at a higher pH, and detecting fluorescence intensity of the cellsafter incubation, wherein an increase in fluorescence compared to acontrol indicates phagocytosis of the POS by the cells.

In some embodiments, the cells are incubated with the POS at atemperature ranging from about room temperature to about 37° C. or aboutroom temperature to about 40° C. In some embodiments, the cells areincubated with the POS at about room temperature, at about physiologicaltemperature, or at about 37° C.

In some embodiments, the control is cells incubated with the POS atbelow room temperature. In some embodiments, the control is cellsincubated with the POS at 4° C.

Also provided herein is a method for assessing phagocytosis activity ofan adherent cell population comprising incubating an adherent cellpopulation with photoreceptor outer segments (POS) for a time andtemperature sufficient for cells in the cell population to phagocytosethe POS, wherein the POS fluoresce more at an acidic pH than at a higherpH, and detecting fluorescence intensity of the cell population afterincubation, wherein an increase in fluorescence compared to a controlindicates phagocytosis of the POS by the cells.

In some embodiments, the adherent cell population is incubated with thePOS at a temperature ranging from about 17-40° C., or from about 25-40°C., or from about 34-40° C., or at a temperature of about 37° C. In someembodiments, the control is a cell population incubated with the POS ata temperature of about 10-16° C. In some embodiments, the control is acell population incubated with the POS at a temperature of about 12-15°C.

Also provided herein is a method for assessing phagocytosis activitycomprising providing photoreceptor outer segments (POS) labeled with afluorescent label having an altered fluorescence signal wheninternalized by phagocytosis into a low pH compartment in a cellrelative to the fluorescence signal when present extracellularly;incubating test cells with the labeled POS under conditions permissivefor the phagocytosis of the labeled POS; and detecting the alteredfluorescence, if any, in the test cells after incubation with thelabeled POS, and quantifying the phagocytosis activity of the test cellstherefrom.

In some embodiments, the altered fluorescence signal (when the label isinternalized by phagocytosis into a low pH compartment) is an increasein intensity of the fluorescence signal relative to the when thefluorescent label is present extracellularly. In some embodiments, thealtered fluorescence signal (when the label is internalized byphagocytosis into a low pH compartment) is detectable by flow cytometry.In some embodiments, the altered fluorescence signal distinguishesbetween labeled POS internalized by phagocytosis and labeled POS boundon the surface of the test cells but not internalized. In someembodiments, the altered fluorescence signal detected in the test cellsis compared to a control cell population incubated with labeled POS inorder to quantify the phagocytosis activity of the test cells.

In some embodiments, the test cells are incubated with labeled POS atabout room temperature, at about physiological temperature, at about 37°C., at about 15-40° C., or between room temperature and 37° C., orbetween room temperature and 40° C.

In some embodiments, the control cell population is incubated withlabeled POS at a below room temperature, including at about 4° C.

Also provided is a method for assessing phagocytosis activity comprisingproviding photoreceptor outer segments (POS) labeled with a fluorescentlabel having an altered fluorescence signal when internalized byphagocytosis into a low pH compartment in a cell relative to thefluorescence signal when present extracellularly; incubating adherenttest cells with the labeled POS under conditions permissive for thephagocytosis of the labeled POS; and detecting the altered fluorescence,if any, in the adherent test cells after incubation with the labeledPOS, and quantifying the phagocytosis activity of the adherent testcells therefrom.

In some embodiments, the test cells are incubated with the POS at atemperature ranging from about 17-40° C., or from about 25-40° C., orfrom about 34-40° C., or at a temperature of about 37° C. In someembodiments, the altered fluorescence signal detected in the adherenttest cells is compared to a control cell population incubated withlabeled POS at a temperature that maintains the viability of the cellsyet induces low or no phagocytosis, optionally such a temperature mayrange from about 12-15° C., in order to quantify the phagocytosisactivity of the test cells. In some embodiments, the control cellpopulation is incubated with the POS at a temperature of about 10-16° C.

Also provided herein is a labeled photoreceptor outer segment (POS)preparation for assessing phagocytic activity of a test cell population,the POS being labeled with a fluorescent label having an alteredfluorescence signal when internalized by phagocytosis into a low pHcompartment in a cell relative to the fluorescence signal when thefluorescent label is present extracellularly. In some embodiments, thealtered fluorescence signal (when the label is internalized byphagocytosis into a low pH compartment) is an increase in intensity ofthe fluorescence signal relative to the when present extracellularly. Insome embodiments, the altered fluorescence signal (when the label isinternalized by phagocytosis into a low pH compartment) is detectable byflow cytometry. In some embodiments, the fluorescent label is pHrodo®Red. In some embodiments, the POS are labeled with pHrodo® Red andpHrodo® Red E. coli Particles.

Also provided herein is a method for measuring phagocytosis activity ina cell population comprising measuring test fluorescence in a test cellpopulation contacted with non-FITC fluorescently labeled photoreceptorouter segments (POS), and comparing the measured test fluorescence to acontrol fluorescence, wherein the non-FITC fluorescently labeled POSfluoresces at an acidic pH but does not fluoresce or minimallyfluoresces at higher pH.

In some embodiments, the test cell population is contacted with non-FITCfluorescently labeled POS at a temperature ranging from about roomtemperature to about physiological temperature, including for exampleabout 37° C. (i.e., about room temperature to about 37° C.), or aboutroom temperature to about 40° C. In some embodiments, the test cellpopulation is contacted with non-FITC fluorescently labeled POS at atemperature between about 15-40° C., or at about physiologicaltemperature, including for example about 37° C. In some embodiments, thecontrol fluorescence is fluorescence of a cell population contacted withthe non-FITC fluorescently labeled POS at below room temperature. Insome embodiments, the control fluorescence is fluorescence of a cellpopulation contacted with the non-FITC fluorescently labeled POS at 4°C.

Also provided herein is a method for measuring phagocytosis activity inan adherent cell population comprising measuring test fluorescence in anadherent test cell population contacted with non-FITC fluorescentlylabeled photoreceptor outer segments (POS), and comparing the measuredtest fluorescence to a control fluorescence, wherein the non-FITCfluorescently labeled POS fluoresces at an acidic pH but does notfluoresce or minimally fluoresces at higher pH.

In some embodiments, the test cell population is contacted with non-FITCfluorescently labeled POS at a temperature ranging from about 17-40° C.,or from about 25-40° C., or from about 34-40° C., or a temperature ofabout 37° C.

In some embodiments, the control fluorescence is fluorescence of anadherent cell population contacted with the non-FITC fluorescentlylabeled POS at temperature of about 12-15° C. In some embodiments, thecontrol fluorescence is fluorescence of an adherent cell populationcontacted with the non-FITC fluorescently labeled POS at temperature ofabout 10-16° C.

Also provided herein is a method for measuring phagocytosis activitycomprising (1) measuring a test fluorescence in a first aliquot of acell population incubated with fluorescently labeled photoreceptor outersegments (POS) that are labeled with pHrodo® Red dye at a temperatureranging from about room temperature to about physiological temperatureincluding for example 37° C., or about room temperature to about 40° C.,and (2) measuring a control fluorescence in a second aliquot of the cellpopulation incubated with fluorescently labeled POS that are labeledwith pHrodo® Red dye at below mom temperature, wherein a testfluorescence that is greater than a control fluorescence indicatesphagocytosis activity of the cell population.

Also provided herein is a method tier measuring phagocytosis activitycomprising (1) measuring a test fluorescence in a first aliquot of anadherent cell population incubated with fluorescently labeledphotoreceptor outer segments (POS) that are labeled with pHrodo® Reddye, and (2) measuring a control fluorescence in a second aliquot of theadherent cell population incubated with fluorescently labeled POS thatare labeled with pHrodo® Red dye, wherein a test fluorescence that isgreater than a control fluorescence indicates phagocytosis activity ofthe cell population.

In some embodiments, the first aliquot of the adherent cell populationis incubated with the labeled POS at a temperature ranging from about17-40° C., or from about 25-40° C., or from about 34-40° C., or atemperature of about 37° C. In some embodiments, second aliquot of theadherent cell population is incubated with the labeled POS at atemperature ranging from about 10-16° C. or 12-15° C.

Also provided herein is a method for measuring phagocytosis activitycomprising (1) measuring a test fluorescence in a first aliquot of acell population incubated with pHrodo® Red labeled photoreceptor outersegments (POS) alone or with pHrodo® Red E. coli BioParticles, at atemperature ranging from about room temperature to about physiologicaltemperature including 37° C., or about room temperature to about 40° C.,and (2) measuring a control fluorescence in a second aliquot of the cellpopulation incubated with pHrodo® Red labeled photoreceptor outersegments (POS) alone or with pHrodo® Red E. coli BioParticles, at belowroom temperature, including 4° C., wherein a test fluorescence that isgreater than a control fluorescence indicates phagocytosis activity ofthe cell population.

Also provided herein is a method for measuring phagocytosis activitycomprising (1) measuring a test fluorescence in a first aliquot of anadherent cell population incubated with pHrodo® Red labeledphotoreceptor outer segments (POS) alone or with pHrodo® Red E. coliBioParticles, at a temperature ranging from about 17-40° C., or fromabout 25-40° C., or from about 34-40° C., or a temperature of about 37°C., and (2) measuring a control fluorescence in a second aliquot of theadherent cell population incubated with pHrodo® Red labeledphotoreceptor outer segments (POS) alone or with pHrodo® Red E. coliBioParticles, at temperature ranging from about 10-16° C. or about12-15° C., wherein a test fluorescence that is greater than a controlfluorescence indicates phagocytosis activity of the cell population.

Various embodiments apply equally to the any and all of theafore-mentioned aspects. These are recited below.

In some embodiments, the cells, cell population, test cells, or testcell population are incubated with the POS at about room temperature, atabout physiological temperature, or at about 37° C., or about roomtemperature to about 40° C., or about room temperature to about 37° C.,or about 15-40° C. In some embodiments, the cells, cell population, testcells, or test cell population are incubated with labeled POS at atemperature ranging from about 17-40° C., or from about 25-40° C., orfrom about 34-40° C., or at a temperature of about 37° C.

In some embodiments, the control cells, control cell population, controltest cells, or control test cell population are incubated with the POSat a temperature that is less than room temperature, a temperatureranging from about 10-16° C. or from about 12-15° C., or a temperatureof about 4° C.

In some embodiments, the cells, cell population, test cells, or testcell population comprise retinal pigmented epithelial (RPE) cells. Insome embodiments, the cells, cell population, test cells, or test cellpopulation comprise photoreceptor progenitor cells. In some embodiments,the cells, cell population, test cells, or test cell population arehuman cells.

In some embodiments, the cells, cell population, test cells, or testcell population are produced by in vitro differentiation of pluripotentstem cells.

In some embodiments, the cells, cell population, test cells, or testcell population were cryopreserved and thawed prior to use.

In some embodiments, the cells, cell population, test cells, or testcell population is provided as a confluent monolayer.

In some embodiments, the cells, cell population, test cells, or testcell population are or is enzyme digested prior to use.

In some embodiments, the cells, cell population, test cells, or testcell population is or are provided as an adherent cell population.

In some embodiments, the POS are fragmented POS. In some embodiments,the POS are sonicated POS.

In some embodiments, the fluorescent label is pHrodo® Red. Thus, in someembodiments, the POS are labeled with pHrodo® Red dye. In someembodiments, the POS are labeled with pHrodo® Red and pHrodo® Red E.coli Bioparticles. In some embodiments, the cells are incubated withpHrodo® Red labeled POS and pHrodo® Red E. coli BioParticles.

In some embodiments, fluorescence is detected by flow cytometry. In someembodiments, fluorescence is detected using a plate reader.

In some embodiments, the cells are incubated with the POS for about15-30 hours, or 16-20 hours, or 20-28 hours.

In some embodiments, the cells are provided as a cell culture. In someembodiments, the cells are a confluent cell culture.

It is to be understood that the test and control cells may be differentaliquots of the same cell population.

In another aspect, this disclosure provides an isolated cell populationcharacterized as having a rate of phagocytosis of photoreceptor outersegments (POS) that is at least 50% greater than a rate of phagocytosisof POS for an equivalent number of primary cells. In some embodiments,the cell population is an RPE cell population. In some embodiments, thecell population is an RPE cell population obtained by in vitrodifferentiation of pluripotent stem cells and the primary cells are RPEcells from isolated adult eyes. In some embodiments, the cell populationis photoreceptor progenitors.

These and various other aspects and embodiments will be described ingreater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. FACS analysis of phagocytosis by RPE. (A) FITC-labeled ROS.(B) pHrodo®-labeled ROS. Shown are the plots for the control in which noROS were added, the control in which ROS were added and the cellsincubated at 4° C., and the test in which ROS were added and the cellsincubated at 37° C. (C) pHrodo® labeled Bioparticles (bacterialfragments). Shown are the plots for the control in which no particleswere added, the control in which particles were added and the cellsincubated at 4° C., and the test in which particles were added and thecells incubated at 37° C. It is to be understood that POS and ROS areused interchangeably herein to refer to the photoreceptor rod outersegments.

FIGS. 2A-B. pH-dependence of fluorescence of FITC (A) and pHrodo® (B)labeled ROS. Plotted is fluorescence at a neutral pH and at an acidicpH.

FIGS. 3A-F. FACS analysis of phagocytosis of fluorescently-labeled ROSby ARPE19 cells in monolayer. No dose-dependent response of phagocytosiswas observed when different concentrations of fluorescently-labeled ROSwere incubated with ARPE-19 for 24 hours in a cell monolayer. (A)ARPE-19 incubated with 6×10⁶ pHrodo® Red-labeled ROS at 37′C. (B)ARPE-19 incubated with 3×10⁶ pHrodo® Red-labeled ROS at 37° C. (C)ARPE-19 incubated with 1.5×10⁶ pHrodo® Red-labeled ROS at 37° C. (D)ARPE-19 incubated with 6×10⁶ pHrodo® Red-labeled ROS at 15° C. (E)ARPE-19 incubated with 3×10⁶ pHrodo® Red-labeled ROS at 15° C. (F)ARPE-19 incubated with 1.5×10⁶ pHrodo® Red-labeled ROS at 15° C. Ineach, plots for cells incubated with no ROS and for cells incubated withROS are shown.

FIGS. 4A-F. FACS analysis of phagocytosis of fluorescently-labeled ROSby hESC-derived RPE cells. No dose-dependent response of phagocytosiswas observed when different concentrations of fluorescently-labeled ROSwere incubated with RPE for 24 hours at 37° C. in a cell monolayer.Pulse sonication of fluorescently-labeled ROS during reconstitutionincreased phagocytosis by approximately 10% (A) RPE cells incubated with3.75×10⁶ pHrodo® Red-labeled ROS reconstituted without sonication. (B)RPE cells incubated with 5×10⁶ pHrodo® Red-labeled ROS reconstitutedwithout sonication. (C) RPE cells incubated with 7.5×10⁶ pHrodo®Red-labeled ROS reconstituted without sonication. (D) RPE cellsincubated with 10×10⁶ pHrodo® Red-labeled ROS reconstituted withoutsonication. (E) RPE cells incubated with 10×10⁶ pHrodo® Red-labeled ROSreconstituted with sonication. (F) RPE cells incubated with 13.5×10⁶pHrodo® Red-labeled ROS reconstituted with sonication.

DETAILED DESCRIPTION

Phagocytosis (potency assay) may be assessed by quantitativefluorescence activated cell sorting (FRCS) analysis of RPE culturesexposed to photoreceptor outer segments (POS) labeled with pHrodo® Reddye (Life Technologies, Molecular Probes).

Phagocytosis may be assessed by a FACS-based assay using POS labeledwith pHrodo® Red dye (Lite Technologies, Molecular Probes). The dye andthe POS so labeled fluoresce when internalized in the reduced pHenvironment of intracellular phagosomes. The POS may be labeled asdescribed herein.

In some embodiments, the RPE cell cultures are confluent. As an example,confluent RPE may be cultured in multiwell plates, and may be incubatedwith POS labeled with pHrodo® Red dye, optionally in the presence ofCO₂-independent medium (Invitrogen). The incubation may occurs for anytime sufficient for the RPE cells to phagocytose the POS. As an example,the incubation may occur for 16-20 hours. The assay occurs at atemperature sufficient for the RPE cells to phagocytose the POS. In someembodiments, the assay occurs at about physiological temperature orabout 37° C. In some embodiments, the assay occurs at room temperature.In some embodiments, the control (or negative control) plates areincubated at 4° C. Cells may be examined under the microscope,fluorescence measured using plate readers, and/or the cells may beharvested after enzyme digestion (e.g., trypsin digestion) and analyzedby flow cytometry.

An exemplary, non-limiting, assay is as follows:

RPE manufactured from pluripotent stem cells (such as but not limited toES cell and iPS cells) as described previously are tested for theirability to phagocytose. The cryopreserved RPE may be previously frozenand thawed prior to use. The RPE cells are seeded in culture in asuitable medium and grown to confluence and maintained in culture priorto testing for their ability to phagocytose POS labeled with pHrodo® Reddye which fluoresces when internalized in the acidic environment ofphagosomes of the RPE cells. RPE cells are incubated with the labeledPOS at 37° C. to permit phagocytosis, or at 4° C. as a negative control.Shifts in fluorescence intensity may be detected by flow cytometry forthe cells incubated at 37° C., indicating phagocytosis of the labeledPOS. Statistical integration of the peaks yield the percentages ofphagocytic positive cells for each lot of RPE cells and incubationtemperature.

As will be understood by this disclosure, phagocytosis is detected byincubating RPE cells with labeled POS that fluoresce in the red spectrumin the acidic phagosome environment. Percentages of phagocytic positivecells may be shown for cells incubated with at 37° C. or at 4° C.(negative control), as detected by flow cytometry.

The resulting RPE cell population may be characterized based on itsphagocytic activity according to the methods provided herein. Rates ofphagocytosis may be determined and the RPE cells so characterized. Forexample, the RPE cells may be characterized as having a rate ofphagocytosis of photoreceptor outer segments (POS) that is at least 50percent greater than the rate of phagocytosis of POS for an equivalentnumber of RPE cells isolated adult eyes, or at least 75, 100, 150 or 200percent greater than the rate of phagocytosis of POS for an equivalentnumber of RPE cells isolated adult eyes. Alternatively or additionally,the RPE cells may be characterized by a rate of phagocytosis ofphotoreceptor outer segments (POS) that is at least 20 percent of thetotal concentration of POS after 24 hours, or at least 25, 30, 25, 40 or50 percent of the total concentration of POS after 24 hours.

Thus, using the methods described herein, RPE cell populations have beenachieved that have a rate of phagocytosis of photoreceptor outersegments (POS) that is at least 54 percent greater than the rate ofphagocytosis of POS for an equivalent number of RPE cells isolated adulteyes (i.e., human adult patients from the age of 25-80, more preferablyadults from the age of 50-80), and more preferably at least 75, 100, 150or even 200 percent greater.

Using the methods described herein, RPE cell populations have beenachieved that have a rate of phagocytosis of photoreceptor outersegments (POS) that is at least 20 percent of the total concentration ofPOS after 24 hours, and more preferably at least 25, 30, 25, 40 or even50 percent of the total concentration of POS after 24 hours.

Thus, this disclosure provides in one aspect a method comprisingdetecting or measuring fluorescence in an RPE cell (or an RPE cellpopulation) contacted with fluorescently labeled photoreceptor outersegments (POS) that are non-FITC fluorescently labeled (regarded as thetest fluorescence, or generally “test”)) and comparing the detected ormeasured fluorescence to a control (regarded as the control fluorescenceor generally “control”). The test may be performed at a temperature thatis about room temperature, or a temperature between about 15-40° C., orat about a physiological temperature (e.g., at about 37° C.). Thecontrol may be performed at about 4° C. Thus, the control fluorescencemay be the fluorescence that is detected or measured followingincubation of RPE cells with non-FITC labeled POS at 4° C. Non-FITCfluorescently labeled POS are POS that are labeled with a fluorophorethat is not FITC. The non-FITC fluorescent label is a label thatfluoresces at an acidic pH such as the pH of the phagosomes, andparticularly phagosomes of RPE cells but that does not fluoresce or thatfluoresces minimally at higher pHs such as neutral pH (or anextracellular environment pH). The non-FITC fluorescent label are usefulin discriminating between surface labeling and internalized labels. Anexample of such a fluorophore is pHrodo® Red dye (Life Technologies,Molecular Probes). A higher degree of phagocytosis in the test isindicated by a higher fluorescence as compared to the control.

Thus, in another aspect, the disclosure provides a method comprising

(1) detecting or measuring fluorescence in a first aliquot of an RPEcell (or an RPE cell population) contacted with (and incubated with)fluorescently labeled photoreceptor outer segments (POS) that arelabeled with pHrodo® Red dye at 37° C. (regarded as the testfluorescence, or generally “test”)), and

(2) detecting or measuring fluorescence in second aliquot of an RPE cell(or an RPE cell population) contacted with (and incubated with)fluorescently labeled photoreceptor outer segments (POS) that arelabeled with pHrodo® Red dye at 4° C. (regarded as the controlfluorescence, or generally “control”)), and

(3) optionally comparing, determining and/or quantifying the test andcontrol fluorescences wherein a test fluorescence that is greater than acontrol fluorescence is an indication of phagocytosis activity of theRPE cell (or cell population).

It is to be understood that the methods described herein may also beused to assay phagocytosis activity in photoreceptor progenitor (orprecursor) cells.

In certain embodiments, the RPE and photoreceptor progenitor cells havephagocytic activity, such as the ability to phagocytose isolated pHrodo®Red photoreceptor outer segments, pHrodo® Red E. coli BioParticles orboth, and the methods provided herein assay one or more of thesefunctions.

In an aspect, the present disclosure provides an assay for determiningthe potency of a pharmaceutical composition comprising: a plurality ofretinal pigment epithelial (RPE) cells or photoreceptor progenitorcells; and a pharmaceutically acceptable carrier. In one embodiment, theaverage melanin content of said plurality of RPE cells is less than 8pg/cell. Said RPE cells or photoreceptor progenitor cells may becontained in a suspension, gel, colloid, matrix, substrate, scaffold, orgraft.

Said pharmaceutically acceptable carrier may comprise a sterile solutionhaving an osmolality of between about 290 mOsm/kg and about 320 mOsm/kg,or between about 300 mOsm/kg and 310 mOsm/kg or about 305 mOsm/kg. Saidpharmaceutically acceptable carrier may comprise a balanced saltsolution. Said balanced salt solution may comprise, consists of, orconsist essentially of, in each mL, sodium chloride 7.14 mg, potassiumchloride 0.38 mg, calcium chloride dihydrate 0.154 mg, magnesiumchloride hexahydrate 0.2 mg, dibasic sodium phosphate 0.42 mg, sodiumbicarbonate 2.1 mg, dextrose 0.92 mg, glutathione disulfide (oxidizedglutathione) 0.184 mg, and hydrochloric acid and/or sodium hydroxide (toadjust pH to approximately 7.4) in water.

The volume of said pharmaceutical composition may be between about 100μL, and 1000 μL or may be at least about 150 μL. Said pharmaceuticalcomposition may comprise between about 1,000 and about 1×10⁹ viable RPEcells. Said pharmaceutical composition may comprise between about 333viable RPE cells/μL and about 2,000 viable RPE cells/μL, between about444 viable RPE cells/μL, and about 1766 viable RPE cells/μL, about 333viable RPE cells/μL, about 444 viable RPE cells/μL, about 666 viable RPEcells/μL, about 888 viable RPE cells/μL, about 999 viable RPE cells/μL,or about 1,333 viable RPE cells/μL.

The concentration of RPE cells in said pharmaceutical composition may besufficiently high that no more than about 30% of said RPE cells loseviability in 60 minutes, and optionally no more than about 10% of saidRPE cells lose viability in 4 hours. Said concentration of RPE cells maybe at least about 1,000 cells/μL, at least about 2,000 cells/μL, betweenabout 1,000-10,000 cells/μL, or between about 2,000-5,000 cells/μL.

The pharmaceutical preparation may comprise less than about 25%, 20%,15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001% cells that maybe not RPE cells.

The average melanin content of said RPE cells may be less than 8pg/cell, less than 7 pg/cell, less than 6 pg/cell, less than 5 pg/cell,less than 4 pg/cell, less than 3 pg/cell, less than 2 pg/cell and atleast 0.1 pg/cell and optionally at least 0.5 pg/cell or 1 pg/cell;between 0.1-8 pg/cell, between 0.1-7 pg/cell, between 0.1-6 pg/cell,between 0.1-5 pg/cell, between 0.1-4 pg/cell, between 0.1-3 pg/cell,between 0.1-2 pg/cell, between 0.1-1 pg/cell, between 1-7 pg/cell,between 0.5-6 pg-cell, or between 1-5 pg/cell.

At least 50%, at least 60%, at least 70%, or at least 80% of the cellsin said pharmaceutical composition may be bestrophin+. At least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% of the cells insaid pharmaceutical composition may be PAX6+ and/or MITF+. At least 80%,at least 85%, at least 90%, at least 95%, or at least 99% of the cellsin said pharmaceutical composition may be PAX6+ and/or bestrophin+. Atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ofthe cells in said pharmaceutical composition may be ZO-1+. At least 50%,at least 60%, or at least 70% of the cells in the pharmaceuticalcomposition may be PAX6+ and bestrophin+. At least 90%, at least 95%, orat least 99% of the cells in said pharmaceutical composition may bePAX6+.

In an exemplary embodiment, no more than about one cell per millioncells and optionally no more than two cells per nine million cells insaid pharmaceutical composition may be positive for both OCT-4 andalkaline phosphatase (AP) expression.

A needle or an injection cannula may contain at least a portion of saidRPE cells. The concentration of said RPE cells upon loading into saidneedle or injection cannula may be between about 444 viable cells/μL andabout 1,766 viable cells/μl. The concentration of viable RPE cells to bedelivered from said needle or injection cannula may be between about 333viable cells/μL and about 1,333 viable cells/μL. The diameter of saidneedle or injection cannula may be between about 0.3 mm and about 0.9.The diameter of said needle or injection cannula may be between about0.5 and about 0.6 mm. Said needle or injection cannula may comprise atip having a diameter between about 0.09 mm and about 0.15 mm. Saidcannula may be a MEDONE POLYTIP® Cannula 25/38 g (a 0.50 mm (25 g)×28 mmcannula with 0.12 mm (38 g)×5 mm tip) or a Synergetics Angled 39 gInjection Cannula.

Said RPE cells may comprise RPE cells which have been cryopreserved andthawed.

Said RPE cells may be human.

Said RPE cells, such as human RPE cells, may be produced from any sourceincluding pluripotent cells such as embryonic stem cells or inducedpluripotent stem cell as well as donor adult or fetal tissue Saidpluripotent stem cell may be positive for expression of one or moremarkers may comprise OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3,SSEA-4, TRA-1-60, and/or TRA-1-81. Said pluripotent cells may be humanpluripotent cells that may be cultured in a multilayer population orembryoid body for a time sufficient for pigmented epithelial cells toappear in said culture. Said time sufficient for pigmented epithelialcells to appear in said culture may comprise at least about 1 week, atleast about 2 weeks, at least about 3 weeks, at least about 4 weeks, atleast about 5 weeks, at least about 6 weeks, or at least about 7 weeks,at least about 8 weeks. Said multilayer population or embryoid body maybe cultured in a medium may comprise DMEM. Said medium may comprise,consists essentially of, or consists of EB-DM. Said pigmented epithelialcells may be isolated and cultured, thereby producing a population ofRPE cells. Said isolating may comprise dissociating cells or clumps ofcells from the culture enzymatically, chemically, or physically andselecting pigmented epithelial cells or clumps of cells may comprisepigmented epithelial cells. Said embryoid body may be cultured insuspension and/or as an adherent culture (e.g., in suspension followedby adherent culture). Said embryoid body cultured as an adherent culturemay produce one or more outgrowths comprising pigmented epithelialcells. Said pluripotent stem cells have reduced HLA antigen complexity.Prior to RPE formation said pluripotent cells may be cultured on amatrix which may be selected from the group consisting of laminin,fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I,collagen IV, collagen VIII, heparan sulfate. Matrigel™ (a solublepreparation from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells),CellStart, a human basement membrane extract, and any combinationthereof. Said matrix may comprise Matrigel™ (a soluble preparation fromEngelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

The pharmaceutical composition may comprise cells that lack substantialexpression of one or more embryonic stem cell markers. Said one or moreembryonic stem cell markers may comprise OCT-4, NANOG, Rex-1, alkalinephosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and/or TRA-1-81.

Said RPE cells may be positive for expression aunt or more RPE cellmarkers. Said one or more RPE cell markers may comprise RPE65, CRALBP,PEDF, Bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and/or tyrosinase.

Said RPE cells may be produced by a method comprising maintaining RPEcells as quiescent cells for a time sufficient to attain said averagemelanin content. Said RPE cells may be produced by a method comprisingmaintaining RPE cells as quiescent cells for a time sufficient toestablish bestrophin expression in at least 50% of said RPE cells.

Said pharmaceutical composition may be substantially free of mouseembryonic feeder cells (MEF) and human embryonic stem cells (hES).

The RPE cells may meet at least one of the criteria recited in Table 1and/or manufactured in accordance with Good Manufacturing Practices(GMP).

Said cryopreserved retinal pigment epithelial (RPE) cells orphotoreceptor progenitors may be provided as a cryopreservedcomposition.

Said RPE cells may exhibit a rate of phagocytosis of photoreceptor outersegments (POS) that may be at least 50 percent greater than the rate ofphagocytosis of POS for an equivalent number of RPE cells from isolatedadult eyes, or at least 75, 100, 150 or 200 percent greater than therate of phagocytosis of POS for an equivalent number of RPE cells fromisolated adult eyes; or a rate of phagocytosis of photoreceptor outersegments (POS) that may be at least 20 percent of the totalconcentration of POS after 24 hours, or at least 25, 30, 25, 40 or 50percent of the total concentration of POS after 24 hours. Saidphotoreceptor progenitors may exhibit a rate of phagocytosis ofphotoreceptor outer segments (POS) that may be at least 50 percentgreater than the rate of phagocytosis of POS for an equivalent number ofphotoreceptor progenitors from isolated adult eyes, or at least 75, 100,150 or 200 percent greater than the rate of phagocytosis of POS for anequivalent number of photoreceptor progenitors from isolated adult eyes;or a rate of phagocytosis of photoreceptor outer segments (POS) that maybe at least 20 percent of the total concentration of POS after 24 hours,or at least 25, 30, 25, 40 or 50 percent of the total concentration ofPOS after 24 hours. The rate and extent of phagocytosis may depend uponthe incubation time and on the maturity of the cells. The rates ofbinding and internalization of POS can be different based on thematurity and pigmentation of the cells. The percentage of cells capableof phagocytosis may be different based on the maturity of the cellcultures.

By labeling the photoreceptor outer segments with a dye which isnon-fluorescent or weakly fluorescent at neutral pH but which becomesmore fluorescent upon acidification, a more sensitive measurement ofinternalized POS in RPE cells or photoreceptor progenitors in aphagocytosis assay is possible. Most of the quantitative methods usedfor phagocytosis assessment (FACS, fluorescence plate reader) do notdiscriminate between internalized and surface bound fluorescentparticles. Additionally, FITC fluorescence is pH-sensitive and issignificantly reduced at pH below t while the pH of lysosomes andphagosomes fused with lysosomes is below 5. Thus fluorescence ofFITC-labeled OS in some instances is not representative of the actualamount of internalized OS. According to its manufacturer (LifeTechnologies, Molecular Probes), pH-sensitive rhodamine-based pHrodo®Red dye is non-fluorescent at neutral pH, that upon acidification turnsbright red. The dye is both fluorogenic and pH-sensitive, and it cantherefore be used as a specific sensor of phagocytic events wherebyacidification of the phagosome following phagocytosis is indicated byred fluorescence.

Use of a dye which is non-fluorescent or weakly fluorescent at neutralpH but becomes more fluorescent upon acidification, such as pHrodo® Redand CypHerSE which is also maximally fluorescent in an acidicenvironment, LysoSensor by Life Technologies dye, to label photoreceptorouter segments is a significant improvement over the prior art methodsand reagents. We used this pH-sensitive rhodamine-based pHrodo® Red dyeto label bovine OS to specifically measure the internalized particles.

When comparing FITC labeled photoreceptor outer segments,pHrodo®-BioParticles and pHrodo®-labeled photoreceptor outer segments,all showed significant increase in fluorescence (FIG. 1A-C) in thephagocytosis assay at 37° C. as compared to 4° C. However, quantitativeanalysis showed that only about half of the RPE cell population at 37°C. in the phagocytosis assay demonstrated increase in fluorescence over4° C. control when incubated with FITC-labeled particles (FIG. 1A), andin the 4° C. control about half of the RAE cell population also showedsignificant increase in fluorescence, possibly indicating the presenceof bound but non-internalized particles on the cell surface. Suchparticles on the cell surface could represent both specific andnon-specific binding. Additionally, interpretation of the FITC-labeledphotoreceptor outer segments FACS data is not very accurate because someof the FITC fluorescence could be lost at low pH (FIG. 2 ), so once theparticles are internalized and phagosomes fuse with lysosomes, at thefinal low pH (4.5-5.5) some of the FITC fluorescence is lost. Thus thefluorescence as measured includes loss of some signal from internalizedparticles and additional signal from non-specifically bound particles onthe surface. Both pHrodo®-labeled BioParticles and pHrodo®-labeledphotoreceptor outer segments did not show any fluorescence increase at4° C. (FIGS. 1B and 1C) but did show an increase at 37° C., thusallowing specific measurement of only internalized particles fused withlysosomes.

As described herein, pHrodo®-labeled ROS become fluorescent at low pH,so an observed shift in fluorescence indicates particles that areinternalized. Use of FITC or other non-pH-sensitive dye-labeled ROStends to show non-specifically bound particles on the cell surface,specifically bound but not internalized POS, and/or POS that isinternalized but not fused with lysosomes. Labeling photoreceptor outersegments with pHrodo® or another pH-sensitive dye, instead ofconventionally used FITC, which fluorescence has an inverse correlationwith the acidity, or using pHrodo®-labeled POS in combination with otherpH-sensitive and/or non-sensitive dye is an improvement in accuracy ofthe phagocytosis assay as it allows for the measurement of phagocytosisof the physiologically relevant target and allows for the dissection ofthe mechanisms of phagocytosis.

In some embodiments, the POS are fragmented prior to use with the cellsof interest. POS may be fragmented using for example sonication, orshearing, or other methods in the art. It has been found, in someinstances, that the fragmented POS result in higher phagocytosisreadings from cells. This may be helpful in distinguishing positiveactivity from control activity.

The phagocytosis assays may be carried out using single cell suspensionof cells or they may be carried out using a monolayer of cells. Thus,the cells may be provided as cell suspensions or they may be provided asa monolayer, including a cultured monolayer. This latter embodiment isuseful, particularly if the cells grow normally as a monolayer, as itallows the phagocytosis activity of the cells to be determined as suchcells would normally exist. The cells may be incubated with the labeledPOS as an adherent layer, such as a monolayer, and then may enzymedigested (e.g., trypsin digested) in order to render them a single cellpopulation which can then be analyzed using for example flow cytometry.

In some embodiments, the cells are incubated with POS at a temperatureof at or above 17° C., or at or above 18° C., or at or above 19° C. orat or above 20° C. The upper limit of the temperature range may be at orbelow 42° C., at or below 41° C., at or below 40° C., at or below 39°C., at or below 38° C., or at or below 37° C. Test phagocytosis activitymay be measured at these temperatures. The cells may be provided as amonolayer, including a cultured monolayer.

In some embodiments, the test cells or test cell population is incubatedwith POS at a temperature ranging from 17-40° C., or from 20-40° C., orfrom 25-40° C., or from 30-40° C., or from 35-40° C. or at a temperatureof about 37° C. The negative control may correspond to cells incubatedwith POS at a temperature ranging from about 4-16° C., 5-16° C., 6-16°C. 7-16° C., 8-16° C., 9-16° C., 10-16° C., 11-16° C., or 12-16° C. Thenegative control may correspond to cells incubated with POS at atemperature ranging from about 4-15° C., 5-15° C., 6-15° C., 7-15° C.,8-15° C., 9-15° C., 10-15° C., 11-15° C., or 12-15° C. The cells may beprovided as a monolayer, including a cultured monolayer.

In some instances, the cells have been previously cryopreserved and arethawed and cultured briefly in order to establish a monolayer. Once inthe monolayer, the phagocytosis activity of the cells may be tested asdescribed herein.

In some embodiments, the cells may be exposed to unlabeled POS for aperiod of time, and then exposed to fluorescently labeled POS to measurephagocytosis for the latter POS. In this way, the cells may be primedprior to the introduction of the labeled POS.

Definitions

In order that the invention herein described may be fully understood,the following detailed description is set forth. Various embodiments ofthe invention are described in detail and may be further illustrated bythe provided examples.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe invention or testing of the present invention, suitable methods andmaterials are described below. The materials, methods and examples areillustrative only, and are not intended to be limiting. The followingterms and definitions are provided herein.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

“Embryonic stem cells” (ES cells), as used herein, refers broadly tocells derived from the inner cell mass of blastocysts or morulae thathave been serially passaged as cell lines. The ES cells may be derivedfrom fertilization of an egg cell with sperm or DNA, nuclear transfer,parthenogenesis, or by means to generate ES cells with homozygosity inthe HLA region. ES cells may also refer to cells derived from a zygote,blastomeres, or blastocyst-staged mammalian embryo produced by thefusion of a sperm and egg cell, nuclear transfer, parthenogenesis, orthe reprogramming of chromatin and subsequent incorporation of thereprogrammed chromatin into a plasma membrane to produce a cell.Embryonic stem cells, regardless of their source or the particularmethod used to produce them, can be identified based on the: (i) abilityto differentiate into cells of all three germ layers, (ii) expression ofat least Oct-4 and alkaline phosphatase, and (iii) ability to produceteratomas when transplanted into immunocompromised animals. The termalso includes cells isolated from one or more blastomeres of an embryo,preferably without destroying the remainder of the embryo (see, e.g.,Chung et al., Cell Stem Cell. 2008 Feb. 7; 2(2):113-7; U.S. PGPub No.20060206953; U.S. PGPub No. 2008/0057041, each of which is herebyincorporated by reference in its entirety). The term also includes cellsproduced by somatic cell nuclear transfer, even when non-embryonic cellsare used in the process. ES cells may be derived from fertilization ofan egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or bymeans to generate ES cells with homozygosity in the HLA region. ES cellsare also cells derived from a zygote, blastomeres, or blastocyst-stagedmammalian embryo produced by the fusion of a sperm and egg cell, nucleartransfer, parthenogenesis, or the reprogramming of chromatin andsubsequent incorporation of the reprogrammed chromatin into a plasmamembrane to produce a cell. Human embryonic stem cells of the presentdisclosure may include, but are not limited to, MA01, MA09, ACT-4, No.3, H1, H7, H9, H14 and ACT30 embryonic stem cells. In certainembodiments, human ES cells used to produce RPE cells are derived andmaintained in accordance with GMP standards.

“Macular degeneration,” as used herein, refers broadly to diseasescharacterized by a progressive loss of central vision associated withabnormalities of Bruch's membrane, the neural retina, and the retinalpigment epithelium. Macular degeneration diseases include but are notlimited to age-related macular degeneration, North Carolina maculardystrophy, Sorsby's fundus dystrophy, Stargardt's disease, patterndystrophy, Best disease, malattia leventinese, Doyne's honeycombchoroiditis, dominant drusen, and radial drusen.

“Pluripotent stem cell,” as used herein, refers broadly to a cellcapable of prolonged or virtually indefinite proliferation in vitrowhile retaining their undifferentiated state, exhibiting a stable(preferably normal) karyotype, and having the capacity to differentiateinto all three germ layers (i.e., ectoderm, mesoderm and endoderm) underthe appropriate conditions.

RPE cell,” “differentiated RPE cell,” “ES derived RPE cell,” and as usedherein, may be used interchangeably throughout to refer broadly to anRAE cell differentiated from a pluripotent stem cell, e.g., using amethods disclosed herein. The term is used generically to refer todifferentiated RPE cells, regardless of the level of maturity of thecells, and thus may encompass RPE cells of various levels of maturity.RPE cells can be visually recognized by their cobblestone morphology andthe initial appearance of pigment. RPE cells can also be identifiedmolecularly based on substantial lack of expression of embryonic stemcell markers such as Oct-4 and NANOG, as well as based on the expressionof RPE markers such as RPE 65, PEDF, CRALBP, and bestrophin. Forexample, a cell may be counted as positive for a given marker if theexpected staining pattern is observed, e.g., PAX6 localized in thenuclei, Bestrophin localized in the plasma membrane in a polygonalpattern (showing localized Bestrophin staining in sharp lines at thecell's periphery), ZO-1 staining present in tight junctions outliningthe cells in a polygonal pattern, and MITF staining detected confined tothe nucleus. Unless otherwise specified, RPE cells, as used herein,refers to RPE cells differentiated in vitro from pluripotent stem cells.

“Mature RPE cell” and “mature differentiated RPE cell,” as used herein,may be used interchangeably throughout to refer broadly to changes thatoccur following initial differentiating of RPE cells. Specifically,although RPE cells can be recognized, in part, based on initialappearance of pigment, after differentiation mature RPE cells can berecognized based on enhanced pigmentation.

“Pigmented,” as used herein refers broadly to any level of pigmentation,for example, the pigmentation that initial occurs when RPE cellsdifferentiate from ES cells. Pigmentation may vary with cell density andthe maturity of the differentiated RPE cells. The pigmentation of a RPEcell may be the same as an average RPE cell after terminaldifferentiation of the RPE cell. The pigmentation of a RPE cell may bemore pigmented than the average RPE cell after terminal differentiationof the RPE cell. The pigmentation of a RPE cell may be less pigmentedthan the average RPE cell after terminal differentiation.

“Photoreceptor progenitor” refers to cells of the neural retina, whichmay be differentiated from embryonic stem cells or induced pluripotentstem cells and that expresses the marker PAX6 while not expressing themarker CHX10 (i.e. CHX10(−)). These cells transiently express CHX10 atretinal neural progenitor stage, but the CHX10 expression is turned offwhen cells differentiate into the photoreceptor progenitor stage. Othermarkers expressed by the photoreceptor progenitors may include: Pax6,Nr2e3, Trβ2, Mash1, RORβ, and NRL. Also, “photoreceptor” may refer topost-mitotic cells differentiated from embryonic stem cells or inducedpluripotent stem cells and that expresses the cell marker rhodopsin orany of the three cone opsins, and optionally express the rod or conecGMP phosphodiesterase. The photoreceptors may also express the markerrecoverin, which is found in photoreceptors. The photoreceptors may berod and/or cone photoreceptors.

Cell Markers: Exemplary cell markers that may be assessed for expressioninclude the following: PAX6, RX 1, SIX3, SIX6, LHX2, TBX3, SOX2, CHX10,Nestin, TRβ2, NR2E3, NRL, MASH1, RORβ, Recoverin, Opsin, Rhodopsin, rodand cone cGMP Phosphodiesterase, which may be assessed at the proteinand/or mRNA (see Fischer A J, Reh T A, Dev Neurosci. 2001;23(4-5):268-76; Baumer et al., Development. 2003 July; 130(13):2903-1 S,Swaroop et al., Nat Rev Neurosci. 2010 August; 11 (8):563-76,Agathocleous and Harris, Annu. Rev. Cell Dev. Biol. 2009.25:45-69, eachof which is hereby incorporated by reference in its entirety). Saidmarker identifiers are generally used as in the literature and in theart, particular in the fields of art in related to the contexts in whichthose gene identifiers are recited herein, which may include literaturerelated to photoreceptors, rods, cones, photoreceptor differentiation,photoreceptor progenitors, neural differentiation, neural stem cells,pluripotent stem cells, and other fields as indicated by context.Additionally, the markers are generally human, e.g., except where thecontext indicates otherwise. The cell markers can be identified usingconventional immunocytochemical methods or conventional PCR methodswhich techniques are well known to those of ordinary skill in the art.

“Signs” of disease, as used herein, refers broadly to any abnormalityindicative of disease, discoverable on examination of the patient; anobjective indication of disease, in contrast to a symptom, which is asubjective indication of disease.

“Symptoms” of disease as used herein, refers broadly to any morbidphenomenon or departure from the normal in structure, function, orsensation, experienced by the patient and indicative of disease.

“Therapy,” “therapeutic,” “treating,” “treat” or “treatment”, as usedherein, refers broadly to treating a disease, arresting or reducing thedevelopment of the disease or its clinical symptoms, and/or relievingthe disease, causing regression of the disease or its clinical symptoms.Therapy encompasses prophylaxis, prevention, treatment, cure, remedy,reduction, alleviation, and/or providing relief from a disease, signs,and/or symptoms of a disease. Therapy encompasses an alleviation ofsigns and/or symptoms in patients with ongoing disease signs and/orsymptoms (e.g., blindness, retinal deterioration.) Therapy alsoencompasses “prophylaxis” and “prevention”. Prophylaxis includespreventing disease occurring subsequent to treatment of a disease in apatient or reducing the incidence or severity of the disease in apatient. The term “reduced”, for purpose of therapy, refers broadly tothe clinical significant reduction in signs and/or symptoms. Therapyincludes treating relapses or recurrent signs and/or symptoms (e.g.,retinal degeneration, loss of vision.) Therapy encompasses but is notlimited to precluding the appearance of signs and/or symptoms anytime aswell as reducing existing signs and/or symptoms and eliminating existingsigns and/or symptoms. Therapy includes treating chronic disease(“maintenance”) and acute disease. For example, treatment includestreating or preventing relapses or the recurrence of signs and/orsymptoms (e.g., blindness, retinal degeneration).

The RPE or photoreceptor progenitor cells of the preparation may have arate of phagocytosis of photoreceptor outer segments (POS) that is atleast 50 percent greater than the rate of phagocytosis of POS for anequivalent number of RPE cells from isolated adult eyes (i.e., humanadult patients from the age of 25-80, more preferably from adults fromthe age of 50-80), and more preferably at least 75, 100, 150 or even 200percent greater. The photoreceptor progenitors of the preparation mayhave a rate of phagocytosis of photoreceptor outer segments (POS) thatis at least 50 percent greater than the rate of phagocytosis of POS foran equivalent number of photoreceptor progenitor cells isolated fromadult eyes (i.e., human adult patients from the age of 25-80, morepreferably adults from the age of 50-80), and more preferably at least75, 100, 150 or even 200 percent greater.

The RPE or photoreceptor progenitor cells of the preparation may have arate of phagocytosis of photoreceptor outer segments (POS) that is atleast 20 percent of the total concentration of POS after 24 hours, andmore preferably at least 25, 30, 25, 40 or even 50 percent of the totalconcentration of POS after 24 hours. POS phagocytosis can be measured,as one illustrative and non-limiting example, using the protocolsdescribed in Bergmann et al. FASEB Journal March 2004 vol. 18 pages562-564, with the variation of the non-FITC labeled POS described herein

The RPE or photoreceptor progenitor cell populations may includedifferentiated RPE cells of varying levels of maturity, or may besubstantially pure with respect to differentiated RPE cells of aparticular level of maturity. The RPE cells may be a substantiallypurified preparation comprising RPE cells of varying levels ofmaturity/pigmentation

Cryopreserved Preparations of RPE Cells

The RPE, cells or photoreceptor progenitors may be stored by anyappropriate method known in the art (e.g., cryogenically frozen) and maybe frozen at any temperature appropriate for storage of the cells. Priorto use of these cells, they may be tested in an assay of the inventionto determine phagocytosis activity and/or potency of the cells.

The RPE cells or photoreceptor progenitor cells that show potency in thephagocytosis assay of the invention may be used for treating retinaldegeneration diseases due to retinal detachment, retinal dysplasia,Angioid streaks, Myopic Macular Degeneration, or retinal atrophy orassociated with a number of vision-altering ailments that result inphotoreceptor damage and blindness, such as, choroideremia, diabeticretinopathy, macular degeneration (e.g., age-related maculardegeneration), retinitis pigmentosa, and Stargardt's Disease (fundusflavimaculatus).

The RPE or photoreceptor progenitor cells provided herein may be humanRPE or photoreceptor progenitor cells. Note, however, that the humancells may be used in human patients, as well as in animal models oranimal patients. For example, the human cells may be tested in mouse,rat, cat, dog, or non-human primate models of retinal degeneration.Additionally, the human cells may be used therapeutically to treatanimals in need thereof, such as in veterinary medicine.

Screening Assays

The disclosure provides a method for identifying agents that modulateRPE cell or photoreceptor progenitor phagocytic activity.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

RPE cells were derived from human embryonic stem cells (hESC) aspreviously described (Klimanskaya et al, 2004) and were used at passages2 through 5 after isolation from pigmented cluster differentiationculture. Cells were cultured in EGM-2 medium (Lonza) until they reachedconfluence and in RPE maintenance medium (Klimanskaya et al, 2004) afterthat Cells used in experiments had established a differentiated RPEphenotype characterized by hexagonal morphology, various levels of brownpigment, cuboidal cell appearance, polarization and tight junctions.Alternatively, cells were used in experiments before they fully maturedbut still after they became confluent.

Bovine Rod Outer Segments (catalog #98740) procured from: InVisionBioresources. FITC Isomer (catalog #F1906) I procured from LifeTechnologies. pHrodo® Red Phagocytosis Particle Labeling Kit (catalog#10026) procured from Life Technologies.

Labeling POS with FITC

Resuspended 1 vial of 10 mg FITC Isomer I to 2 mg/mL in 0.1 M SodiumCarbonate Buffer, pH 9.5. Spun down at 3000 g for 10 minutes to removeundiluted particles and only used supernatant for labeling bovine ROS.Thawed 25 bovine eyeballs worth of ROS and resuspended in 5 mL washbuffer (10% sucrose in 20 mM phosphate buffer with 5 mM taurine, pH7.2). Added 1.5 mL of 2 mg/mL. FITC supernatant to the resuspended ROSand let them incubate in dark with rocking for 1 hr at RT. Afterincubation, spun ROS-FITC segments down at 3000 g and resuspended in 10mL wash buffer. Repeated this washing step a total of 2 times. Afterwashing, resuspended in 10 mL 2.5% sucrose in DMEM (Gibco #11960) andspun down again at 3000 g for 10 minutes. Finally, resuspended cells in10 mL 2.5% sucrose in DMEM, counted ROS-FITC particles using ahemacytometer, adjusted concentration to 1*10⁸ particles/mL in 2.5%sucrose in DMEM, and froze down particles 80° C.

Labeling POS with pHrodo®

Resuspended 25 bovine eyes worth of ROS in 4.165 mL 0.1 M SodiumBicarbonate buffer from pHrodo® Red phagocytosis Particle Labeling kit.Aliquoted ROS out into 4 750 μL aliquots in microcentrifuge tubes.Centrifuged tubes at 10000 RPM for 1 minute, and then resuspended in 750μL 0.1 M Sodium Bicarbonate buffer again. Resuspended pHrodo® dye to afinal concentration of 10 mM in DMSO. Added pHrodo® dye to ROS in sodiumbicarbonate buffer to a final concentration of 0.5 mM, and incubated inthe dark for 45 minutes. After 45 minutes, added 500 μL “Component C”(from kit) and centrifuged at 10000 RPM for 1 minute. Aspiratedsupernatant and resuspended in 1 mL 100% methanol. Vortexed tubes for 30seconds and spun down at 10000 RPM for 1 minute. Aspirated methanol andresuspended in 1 mL “Component C” (wash buffer from kit) and centrifugedagain at 10000 RPM for 1 minute. Repeated this wash step a total of 2times. Resuspended all particles in a total of 20 mL “Buffer B” (fromkit). Spun ROS-pHrodo® down at 3000 RPM for 10 minutes, resuspendedROS-pHrodo® in 2.5% sucrose in DMEM, adjusted concentration to 1*10⁸particles/mL in 2.5% sucrose in DMEM, and froze down particles at −80°C.

RPE cells were incubated with either pHrodo®-conjugated BioParticles orwith bovine outer segments labeled with either FITC or pHrodo® forvarious time from 2 to 24 h at 37° C. After that the cells were washed,harvested by trypsin/dissociation buffer, 1:1, centrifuged and analyzedby flow cytometry. As a negative control, cells were incubated for thesame length of time at 4° C.

Additionally, interpretation of the FITC-Labeled ROS FACS data is notvery accurate because some of the FITC fluorescence could be lost at lowpH (FIGS. 2A and B), so once the particles are internalized andphagosomes fuse with lysosomes, the final low pH (4.5-5.5) some of theFITC fluorescence is lost. Thus the fluorescence as measured includesloss of some signal from internalized particles and additional signalfrom non-specifically bound particles on the surface.

pHrodo® is pH-sensitive fluorescent dye, and both pHrodo®-labeledBioParticles and ROS did not show any fluorescence increase at 4° C.(FIGS. 1B and 1C) thus allowing to specifically measure onlyinternalized particles fused with lysosomes.

Labeling ROS with pHrodo® is an improvement in accuracy of thephagocytosis assay and can be used instead of or complementary toFITC-labeled ROS to dissect the complex mechanisms of phagocytosis.

Labeling with pHrodo® E. coli Fluorescent Bioparticles

Phagocytosis is assessed by a FACS-based assay using pHrodo® E. colifluorescent bioparticles (Invitrogen) which fluoresce when internalizedin the reduced pH environment of intracellular phagosomes. Bioparticleswere prepared according to the manufacturer's instructions. ConfluentRPE were incubated with 50-200 μL bioparticles per one well of a 4-wellplate in CO₂-independent medium (Invitrogen) for 16-20 hours at 37° C.Negative control plates were incubated at 4° C. Cells were examinedunder the microscope, harvested by trypsin and analyzed by FACS counting10,000 events on a C6 Flow Cytometer.

TABLE 1 RPE Cell Characterization and Safety Testing Test SpecificationTest lot Sterility Negative Negative Mycoplasma Negative Negative Celldensity 1-2 million viable cells/mL 2 × 10⁶ (post dilution) viablecells/mL Cell viability Final harvest: >85%  99% Post-thaw: >70%  95%Morphology Confluent, cobblestone Pass epithelium, medium pigmentationKaryotype 46, XX, normal 46, XX, normal DNA fingerprinting Conforms withhESC MCB Conforms hRPE mRNA for: Up-regulated by a minimum RPE-6 1.32BEST-1 of 1 log₁₀ compared to PAX6 2.80 RPE-65 hESC MITF 2.89 PAX6BEST-1 3.81 MITF hESC mRNA for: OCT-4 Down-regulated compared to OCT-4−3.18 NANOG hESC (log₁₀): NANOG −2.49 SOX-2 OCT-4 ≤ −2.13 SOX-2 −2.07NANOG ≤ −1.95 SOX-2 ≤ −0.63 Maturity by bestrophin >70% staining  71%staining Purity by >95% PAX6 and/or MITF 100% immunostaining >95% PAX6and/or 100% bestrophin >95% ZO-1 100% hESC protein markers <2 cellsstaining with 0 OCT-4 and AP in 9 million cells examined Residual murineDNA Negative Negative Murine viruses by MAP Negative NegativeRetroviruses by Negative Negative Mus dunni co-cultivation Ecotropicmurine viruses Negative Negative Endotoxin <0.50 EU/mL 0.312 EU/mLPotency by phagocytosis Positive PositivePhagocytosis of pHrodo® Red-Labeled ROS by RPE Cells.

hESC-derived RPE and ARPE-19 cells were cultured in RPE Growth Medium(RPE-GM) consisting of Endothelial Cell Growth Medium (Lonza, cat#CC-3162, CC-3156).

For the phagocytosis assay, RPE cells were seeded in 96-well cultureplates (Berton-Dickinson) at a density of 5×10⁵ cells/cm² and maintainedin a humidified incubator at 37° C. with 5% CO₂. For optimal assayreadings, RPE cells were cultured for 3-5 days in RPE-GM prior toevaluation of phagocytosis capability. If cells were cultured longerthan 5 days, RPE-GM was switched to RPE Maintenance Medium (RPE-MM)consisting of DMEM supplemented with 10% Fetal Bovine Serum, GlutaMaxand Normocin. The medium was changed every 2-3 days to providesufficient nutrition. For the determination of phagocytotic activitiesof RPE cells, pH-sensitive Rhodamine-based pHrodo® Red-labeled Rod OuterSegments (ROS) (InVision Bioresources, cat. #98740) were used. ROSlabeling with pHrodo® Red Microscale Labeling Kit (Thermo FisherScientific, cat. #P35363) was previously described. Each well ofconfluent RPE cells was inoculated with 0.1 mL DMEM medium containing10% FBS and Normocin and 0.1 mL labeled ROS, reconstituted in DMEMsupplemented with 10% FBS and Normocin. To evaluate optimal phagocytoticcapability of RPE cells, different ROS concentrations of 1.5×10⁶, 3×10⁶,3.75×10⁶, 5×10⁶, 6×10⁶, 7.5×10⁶, 10×10⁶ and 13.5×10⁶ ROS/well of a96-well plate were tested. To decrease ROS aggregates and increasephagocytosis efficiency, a direct pulse-sonication step was introducedduring reconstitution of Rod Outer Segments. RPE cells and ROS wereincubated for 20 to 28 hours at 37° C. for the test samples and 12-15°C. for the negative control in an atmosphere of 5% CO₂ and 95% air. Nextday, after 20 to 28 hours incubation of ROS with RPE cell monolayer,culture medium was aspirated and each well was washed 3× with 0.2 mLCa/Mg-free PBS (Gibco/Invitrogen #14190-250). 0.2 mL of 0.25%Trypsin/EDTA (Sigma, cat. #T4049) plus Cell Dissociation Buffer(Gibco/Invitrogen, cat. #13151) was added to each well in a 1:1 ratioand incubated at room temperature until a single cell suspension wasvisible (10-20 minutes). Cell suspension of each sample was transferredto appropriately labeled round-bottom, polystyrene tube containing 2 ml.DMEM plus 10% FBS to neutralize reaction and centrifuged at 160 g for 5minutes. Supernatant was decanted by leaving ˜0.2-0.25 mL liquid behind.Sample tubes were vortexed and ROS uptake by RPE cells was evaluatedusing BD Accuri C6 Flow Cytometer as previously described.

To further increase phagocytotic capability, naïve RPE cells inmonolayer can be rendered “competent” by exposure to unlabeled ROS fordefined periods of time with or without recovery steps prior toperforming phagocytosis of pHrodo® Red-labeled ROS following the abovedescribed procedure.

REFERENCES

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What is claimed is:
 1. A method of determining whether a cell populationis suitable for treating a retinal degeneration disease, comprising: a)conducting a phagocytosis assay involving the cell population andphotoreceptor outer segments (POS) and/or bacterial fragments; and b)analyzing a rate of phagocytosis of the POS and/or bacterial fragmentsby the cell population, wherein the cell population is suitable fortreating a retinal degeneration disease when: (i) the rate ofphagocytosis of the POS and/or bacterial fragments by the cellpopulation is at least 50 percent greater than the rate of phagocytosisof POS and/or bacterial fragments by an equivalent number of retinalpigment epithelial (RPE) or photoreceptor progenitor cells isolated fromadult eyes, and/or (ii) the cell population phagocytoses at least 20percent of the total concentration of POS and/or bacterial fragmentsafter 24 hours, wherein the cell population is a RPE cell population ora photoreceptor progenitor cell population produced by in vitrodifferentiation of pluripotent stem cells.
 2. The method of claim 1,wherein the cell population is suitable for treating a retinaldegeneration disease when the rate of phagocytosis of the POS and/orbacterial fragments is at least 75, 100, 150 or 200 percent greater thanthe rate of phagocytosis of POS and/or bacterial fragments for anequivalent number of RPE cells or photoreceptor progenitor cells fromisolated adult eyes.
 3. The method of claim 1, wherein the cellpopulation is suitable for treating a retinal degeneration disease whenthe cell population phagocytoses at least 25, 30, 40 or 50 percent ofthe total concentration of POS and/or bacterial fragments after 24hours.
 4. The method of claim 1, wherein the phagocytosis assaycomprises the steps of: incubating the cell population with the POSand/or bacterial fragments for a time and at a temperature sufficientfor cells in the population to phagocytose the POS and/or bacterialfragments, wherein the POS and/or bacterial fragments are labeled with afluorescent label that fluoresces more at an acidic pH than at a higherpH, and detecting fluorescence intensity of the cell population afterincubation, wherein an increase in fluorescence intensity compared to acontrol indicates phagocytosis of the POS and/or bacterial fragments bythe cell population, wherein the cell population is incubated with thePOS and/or bacterial fragments at a temperature ranging from 25-40° C.,or 34-40° C., or at about 37° C., and wherein the control is cellsincubated with the POS and/or bacterial fragments at a temperatureranging from 10-16° C. or 12-15° C.
 5. The method of claim 1, whereinthe phagocytosis assay comprises the steps of: incubating the cellpopulation with the POS for a time and at a temperature sufficient forcells in the population to phagocytose the POS, wherein the POS arelabeled with a fluorescent label that fluoresces more at an acidic pHthan at a higher pH, and detecting fluorescence intensity of the cellpopulation after incubation, wherein an increase in fluorescenceintensity compared to a control indicates phagocytosis of the POS by thecell population, wherein the cell population is incubated with the POSat a temperature ranging from 25-40° C., or 34-40° C., or at about 37°C., and wherein the control is cells incubated with the POS at atemperature ranging from 10-16° C. or 12-15° C.
 6. The method of claim4, wherein the POS and/or bacterial fragments are labeled with afluorescent label having an increased fluorescence signal wheninternalized by phagocytosis into a low pH compartment in a cellrelative to its fluorescence signal when present extracellularly; andwherein the method comprises detecting fluorescence in the cellpopulation after incubation with the labeled POS and/or bacterialfragments relative to control, and quantifying phagocytosis activity ofthe cell population.
 7. The method of claim 4, wherein the fluorescenceis detected by flow cytometry or using a plate reader.
 8. The method ofclaim 4, wherein the cell population is incubated with the POS and/orbacterial fragments at a temperature ranging from 34-40° C. or at about37° C.
 9. The method of claim 4, wherein the control is cells incubatedwith the POS and/or bacterial fragments at a temperature ranging from12-15° C.
 10. The method of claim 4, wherein the cell population and thecontrol cells are incubated with the POS and/or bacterial fragments forabout 16-20 hours.
 11. The method of claim 1, wherein the cellpopulation lacks expression of one or more embryonic stem cell markersselected from the group consisting of OCT-4, NANOG, Rex-1, alkalinephosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. 12.The method of claim 1, wherein the cell population is an RPE cellpopulation that expresses one or more RPE cell markers selected from thegroup consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, Otx2, PAX2,PAX6, ZO-1, and tyrosinase.
 13. The method of claim 1, wherein theretinal degeneration disease is selected from the group consisting ofretinal detachment, retinal dysplasia, angioid streaks, retinal atrophy,choroideremia, diabetic retinopathy, macular degeneration, age relatedmacular degeneration, myopic macular degeneration, retinitis pigmentosa,and Stargardt's Disease (fundus flavimaculatus).
 14. The method of claim1, wherein the bacterial fragments are bioparticles.
 15. The method ofclaim 1, wherein the POS are fragmented POS or sonicated POS.
 16. Themethod of claim 1, wherein the POS and/or bacterial fragments arelabeled with pH-sensitive rhodamine-based dye.
 17. The method of claim1, wherein the RPE cells are human RPE cells or wherein thephotoreceptor progenitor cells are human photoreceptor progenitor cells.18. A method of determining whether a cell population is suitable fortreating a retinal degeneration disease, comprising: incubating a cellpopulation with photoreceptor outer segments (POS) and/or bacterialfragments for a time and temperature sufficient for cells in thepopulation to phagocytose the POS and/or bacterial fragments, whereinthe POS and/or bacterial fragments are labeled with a fluorescent labelthat fluoresces more at an acidic pH than at a higher pH, and detectingfluorescence intensity of the cell population after incubation, whereinan increase in fluorescence intensity compared to a control indicatesphagocytosis of the POS and/or bacterial fragments by the cells andfurther indicates that the cell population is suitable for treating aretinal degeneration disease, wherein the cell population is incubatedwith the POS and/or bacterial fragments at a temperature ranging from25-40° C., or 34-40° C., or at about 37° C., and wherein the control iscells incubated with the POS and/or bacterial fragments at a temperatureranging from 10-16° C. or 12-15° C.
 19. The method of claim 1, furthercomprising assessing proliferative potential of the cell population. 20.The method of claim 19, wherein a proliferative potential that isgreater than the proliferative potential of cells derived from eyedonors indicates that the cell population is suitable fortransplantation.